Ruey-Hwa Chen

TGF-β induces apoptosis through Smad-mediated expression of DAP-kinase

Transforming growth factor-β (TGF-β) and TGF-β-related factors induce apoptosis in a variety of tissues; however, the mechanism underlying this induction is largely unknown. Here, we demonstrate that TGF-β induces the expression of the death-associated protein kinase (DAP-kinase) as an immediate early response in cells that undergo apoptosis in response to TGF-β. DAP-kinase is a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that the DAP-kinase promoter is activated by TGF-β through the action of Smad2, Smad3 and Smad4. Overexpression of DAP-kinase triggers apoptosis in the absence of TGF-β, whereas inhibition of DAP-kinase activity protects cells from TGF-β-induced apoptosis, blocks TGF-β-induced release of cytochrome c from mitochondria and prevents TGF-β-induced dissipation of the mitochondrial membrane potential. Our findings indicate that DAP-kinase mediates TGF-β-dependent apoptosis by linking Smads to mitochondrial-based pro-apoptotic events.

Bidirectional signals transduced by DAPK–ERK interaction promote the apoptotic effect of DAPK

Death‐associated protein kinase (DAPK) is a death domain‐containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify the extracellular signal‐regulated kinase (ERK) as a DAPK‐interacting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser 735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. In a physiological apoptosis system where ERK–DAPK interplay is reinforced, downregulation of either ERK or DAPK suppresses such apoptosis. These results indicate that bidirectional signalings between DAPK and ERK may contribute to the apoptosis‐promoting function of the death domain of DAPK.

miR-103/107 Promote Metastasis of Colorectal Cancer by Targeting the Metastasis Suppressors DAPK and KLF4

Metastasis is the major cause of poor prognosis in colorectal cancer (CRC), and increasing evidence supports the contribution of miRNAs to cancer progression. Here, we found that high expression of miR-103 and miR-107 (miR-103/107) was associated with metastasis potential of CRC cell lines and poor prognosis in patients with CRC. We showed that miR-103/107 targeted the known metastasis suppressors death-associated protein kinase (DAPK) and Krüppel-like factor 4 (KLF4) in CRC cells, resulting in increased cell motility and cell-matrix adhesion and decreased cell-cell adhesion and epithelial marker expression. miR-103/107 expression was increased in the presence of hypoxia, thereby potentiating DAPK and KLF4 downregulation and hypoxia-induced motility and invasiveness. In mouse models of CRC, miR-103/107 overexpression potentiated local invasion and liver metastasis effects, which were suppressed by reexpression of DAPK or KLF4. miR-103/107-mediated downregulation of DAPK and KLF4 also enabled the colonization of CRC cells at a metastatic site. Clinically, the signature of a miR-103/107 high, DAPK low, and KLF4 low expression profile correlated with the extent of lymph node and distant metastasis in patients with CRC and served as a prognostic marker for metastasis recurrence and poor survival. Our findings therefore indicate that miR-103/107-mediated repression of DAPK and KLF4 promotes metastasis in CRC, and this regulatory circuit may contribute in part to hypoxia-stimulated tumor metastasis. Strategies that disrupt this regulation might be developed to block CRC metastasis.

DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals.

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.

Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking src to STAT3 activation

ABSTRACT Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin.

ABSTRACT Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.

A Cullin3-KLHL20 Ubiquitin Ligase-Dependent Pathway Targets PML to Potentiate HIF-1 Signaling and Prostate Cancer Progression

Tumor hypoxia is associated with disease progression and treatment failure, but the hypoxia signaling mechanism is not fully understood. Here, we show that KLHL20, a Cullin3 (Cul3) substrate adaptor induced by HIF-1, coordinates with the actions of CDK1/2 and Pin1 to mediate hypoxia-induced PML proteasomal degradation. Furthermore, this PML destruction pathway participates in a feedback mechanism to maximize HIF-1α induction, thereby potentiating multiple tumor hypoxia responses, including metabolic reprogramming, epithelial-mesenchymal transition, migration, tumor growth, angiogenesis, and chemoresistance. In human prostate cancer, overexpression of HIF-1α, KLHL20, and Pin1 correlates with PML down-regulation, and hyperactivation of the PML destruction pathway is associated with disease progression. Our study indicates that the KLHL20-mediated PML degradation and HIF-1α autoregulation play key roles in tumor progression.

The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and possesses apoptotic and tumor-suppressive functions. However, it is unclear whether DAPK elicits apoptosis-independent activity to suppress tumor progression. We show that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These effects are mainly mediated by an inhibitory role of DAPK in talin head domain association with integrin, thereby suppressing the integrin–Cdc42 polarity pathway. We present evidence indicating that the antimigratory effect of DAPK represents a mechanism through which DAPK suppresses tumors. First, DAPK can block migration and invasion in certain tumor cells that are resistant to DAPK-induced apoptosis. Second, using an adenocarcinoma cell line and its highly invasive derivative, we demonstrate DAPK level as a determining factor in tumor invasiveness. Collectively, our study identifies a novel function of DAPK in regulating cell polarity during migration, which may act together with its apoptotic function to suppress tumor progression.

The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses

Death‐associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)‐induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB–Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch‐repeat domain and BTB domain, respectively. The KLHL20–Cul3–ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20‐mediated DAPK ubiquitination is suppressed in cells receiving IFN‐α or IFN‐γ, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN‐induced autophagy. This study identifies KLHL20–Cul3–ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN‐induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.

Breast Tumor Kinase Phosphorylates p190RhoGAP to Regulate Rho and Ras and Promote Breast Carcinoma Growth, Migration, and Invasion

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.